Targeted antitumor prodrug therapy using CNGRC-yCD fusion protein in combination with 5-fluorocytosine. Issue 1 (December 2016)

• Record Type: Journal Article
• Authors: Li, Jia-Je; Chang, Shun-Fu; Liau, I-Iu; Chan, Pei-Chia; Liu, Ren-Shyan; Yen, Sang-Hue; Wang, Hsin-Ell; Chang, Cheng
• Is Part Of: Journal of biomedical science. Volume 23:Issue 1(2016)
• Page Start: 1
• Page End: 10
• Publication Date: 2016-12
• Subjects: Targeted cancer therapy -- Aminopeptidase N -- Asparagine-glycine-arginine motif -- Cytosine deaminase -- 5-fluorocytosine -- 5-fluorouracil; Medical sciences -- Periodicals; 610.28
• DOI: 10.1186/s12929-016-0227-6
• Languages: English
• ISSNs: 1021-7770

Abstract

Abstract Background The enzyme-prodrug system is considered a promising tool for tumor treatment when conjugated with a targeting molecule. The asparagine-glycine-arginine (NGR) motif is a developing and interesting targeting peptide that could specifically bind to aminopeptidase N (APN), which is an NGR receptor expressed on the cell membrane of angiogenic endothelial cells and a number of tumor cells within the tumor tissues. The objective of this study was to develop a novel targeted enzyme-prodrug system using 5-fluorocytosine (5-FC) and an NGR-containing peptide fused with yeast cytosine deaminase (yCD), i.e. CNGRC-yCD fusion protein, to target APN-expressing cells within the tumor tissues and to convert 5-FC into 5-fluorouracil (5-FU) to kill tumors. Results Both yCD and CNGRC-yCD proteins were cloned into the pET28a vector and expressed by anEscherichia coli host. Both yCD and CNGRC-yCD proteins were purified and the yields were approximately 20 mg/L with over 95 % purity. The binding assay demonstrated that the CNGRC-yCD fusion protein had specific binding affinity toward purified APN recombinant protein and high-APN-expressing cells, including human endothelial cells (HUVECs) and various types of human tumor cell lines, but not low-APN-expressing tumor cell lines. Moreover, the enzyme activity and cell viability assay showed that the CNGRC-yCD fusion protein could effectively convert 5-FC into 5-FU and resulted in significant cell death in both high-APN-expressing tumor cells and HUVECs. Conclusions This study successfully constructs a new targeting enzyme-prodrug system, CNGRC-yCD fusion protein/5-FC. Systematic experiments demonstrated that the CNGRC-yCD protein retained both the APN-binding affinity of NGR and the enzyme activity of yCD to convert 5-FC into 5-FU. The combined treatment of the CNGRC-yCD protein with 5-FC resulted in the significantly increased cell death of high-APN-expressing cells as compared to that of low-APN-expressing cells.